The following procedure is provided for the determination of vitamin A as an ingredient of Pharmacopeial preparations. It conforms to that which was adopted in 1956 for international use by the International Union of Pure and Applied Chemistry.

Complete the assay promptly, and exercise care throughout the procedure to keep to a minimum the exposure to actinic light and to atmospheric oxygen and other oxidizing agents, preferably by the use of low-actinic glassware and an atmosphere of an inert gas.

Special Reagents

ETHER
Use ethyl ether, and use it within 24 hours after opening the container.

Procedure
Accurately weigh, count, or measure a portion of the test specimen expected to contain the equivalent of not less than 0.15 mg of retinol but containing not more than 1 g of fat. If in the form of capsules, tablets, or other solid, so that it cannot be saponified efficiently by the ensuing instructions, reflux the portion taken in 10 mL of water on a steam bath for about 10 minutes, crush the remaining solid with a blunt glass rod, and warm for about 5 minutes longer.

Transfer to a suitable borosilicate glass flask, and add 30 mL of alcohol, followed by 3 mL of potassium hydroxide solution (9 in 10). Reflux in an all-borosilicate glass apparatus for 30 minutes. Cool the solution, add 30 mL of water, and transfer to a conical separator. Add 4 g of finely powdered sodium sulfate decahydrate. Extract by shaking with one 150-mL portion of ether for 2 minutes, and then, if an emulsion forms, with three 25-mL portions of ether. Combine the ether extracts, if necessary, and wash by swirling gently with 50 mL of water. Repeat the washing more vigorously with three additional 50-mL portions of water. Transfer the washed ether extract to a 250-mL volumetric flask, add ether to volume, and mix.

Evaporate a 25.0-mL portion of the ether extract to about 5 mL. Without applying heat and with the aid of a stream of inert gas or vacuum, continue the evaporation to about 3 mL. Dissolve the residue in sufficient isopropyl alcohol to give an expected concentration of the equivalent of 3 µg to 5 µg of vitamin A per mL or to give an absorbance in the range 0.5 to 0.8 at 325 nm. Determine the absorbances of the resulting solution at the wavelengths 310 nm, 325 nm, and 334 nm, with a suitable spectrophotometer fitted with matched quartz cells, using isopropyl alcohol as the blank.

WHEN TOCOPHEROL IS PRESENT
Transfer to a suitable borosilicate glass flask a test specimen, accurately measured, or not less than 5 previously crushed capsules or tablets. Reflux in an all-borosilicate glass apparatus with 30 mL of alcohol and 3 mL of potassium hydroxide solution (9 in 10) for 30 minutes. Add through the condenser 2.0 g of citric acid monohydrate, washing the walls of the condenser with 10 mL of water. Cool, and transfer the solution to a conical separator with the aid of 20 mL of water. Add 4 g of finely powdered sodium sulfate decahydrate. Extract with one 150-mL portion of ether and then, if an emulsion forms, with three 25-mL portions of ether. Combine the ether extracts, if necessary, and wash by swirling gently with 50 mL of water. Repeat the washing more vigorously with three additional 50-mL portions of water. Transfer the washed ether extract to a 250-mL volumetric flask, and add ether to volume. Transfer a 100.0-mL aliquot of the resulting ether solution to a conical separator, and wash once with 50 mL of potassium hydroxide solution (1 in 33), using alcohol, if necessary, to break any emulsion that forms. Wash by swirling gently with 50 mL of water. Repeat the washing more vigorously with three additional 50-mL portions of water. Transfer the washed ether extract to a 100-mL volumetric flask, add ether to volume, and mix.

Evaporate a 50.0-mL aliquot of the ether solution of the unsaponifiable extract to about 5 mL. Without applying heat and with the aid of a stream of inert gas or vacuum, remove the residual ether. Dissolve the residue in 50.0 mL of isopropyl alcohol.

Hydrogenated Portion
Pipet 15.0 mL of the isopropyl alcohol solution into a 50-mL centrifuge tube, add approximately 200 mg of palladium catalyst, stir with a glass rod, and hydrogenate for 10 minutes in a Hydrogenator such as is described in the Alpha Tocopherol Assay 551, using isopropyl alcohol in the blank tube. Add about 300 mg of chromatographic siliceous earth, stir with a glass rod, and immediately centrifuge until the solution is clear.

Test a 1-mL aliquot of the solution by removing the solvent by evaporation, dissolving the residue in 1 mL of chloroform, and adding 10 mL of phosphomolybdic acid TS: no detectable blue-green color appears. [NOTEIf a blue-green color appears, repeat the hydrogenation for a longer time period, or with a new lot of catalyst.]

Into two separate flasks pipet equal volumes of the Hydrogenated Portion and the untreated isopropyl alcohol solution, respectively, and add sufficient isopropyl alcohol to give an expected concentration of vitamin A equivalent to 3 µg to 5 µg per mL. Determine the absorbances of the untreated solution against the solution from the Hydrogenated Portion as a blank, at the wavelengths 310 nm, 325 nm, and 334 nm, with a suitable spectrophotometer fitted with matched quartz cells.

Calculation
Calculate the vitamin A content as follows:

Content (in mg) = 0.549A325 / LC,

in which A325 is the observed absorbance at 325 nm; L is the length, in cm, of the absorption cell; and C is the amount of test specimen expressed as g, capsule, or tablet in each 100 mL of the final isopropyl alcohol solution, provided that A325 has a value not less than [A325]/1.030 and not more than [A325]/0.970, where [A325] is the corrected absorbance at 325 nm and is given by the equation:

[A325] = 6.815A325  2.555A310  4.260A334,

in which A designates the absorbance at the wavelength indicated by the subscript.

Where [A325] has a value less than A325/1.030, apply the following equation:

Content (in mg) = 0.549[A325] / LC,

in which the values are as defined herein. Each mg of vitamin A (alcohol) represents 3333 USP Units of vitamin A.

Confidence Interval
The range of the limits of error, indicating the extent of discrepancy to be expected in the results of different laboratories at P = 0.05, is approximately ±8%.

CHROMATOGRAPHIC METHOD

The following pressurized liquid chromatographic procedure is provided for the determination of Vitamin A. Where the use of vitamin A ester (retinyl acetate or retinyl palmitate) is specified in the following procedure, use the chemical form present in the raw material. Use low-actinic glassware throughout this procedure.

System Suitability Preparation
Dissolve an accurately weighed quantity of retinyl palmitate and USP Vitamin A RS in n-hexane to obtain a solution containing about 7.5 µg per mL of each.

Standard Preparation
Dissolve an accurately weighed quantity of USP Vitamin A RS in n-hexane, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 15 µg of retinyl acetate per mL.

Assay Preparation
Transfer about 15 mg of vitamin A ester (retinyl acetate or retinyl palmitate), accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with n-hexane to volume, and mix. Pipet 5.0 mL of this solution into a 50-mL volumetric flask, dilute with n-hexane to volume, and mix.

Chromatographic System
(see Chromatography 621)The liquid chromatograph is equipped with a 325-nm detector and a 4.6-mm × 15-cm column that contains packing L8. The flow rate is about 1 mL per minute. Chromatograph the System Suitability Preparation, and record the peak responses as directed for Procedure: the resolution, R, between retinyl acetate and retinyl palmitate is not less than 10; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure
Separately inject equal volumes (about 40 µL) of the Standard Preparation and the Assay Preparation into the chromatograph, record the chromatograms, and measure the responses for retinyl acetate obtained from the Standard Preparation and the peak area for retinyl acetate or retinyl palmitate in the chromatogram of the Assay Preparation. Calculate the quantity, in mg, of vitamin A as the retinol equivalent (C20H30O) in the portion of vitamin A taken by the formula:

0.872CD(rU / rS),

in which 0.872 is the factor used to convert retinyl acetate, obtained from USP Vitamin A RS to its retinol equivalent; C is the concentration, in mg per mL, of USP Vitamin A RS in the Standard Preparation; D is the dilution factor, in mL, for the Assay Preparation; and rU and rS are the peak responses of the retinyl ester obtained from the Assay Preparation and the Standard Preparation, respectively. [NOTEThe molar responses of retinyl acetate and retinyl palmitate are equivalent.]